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Serine/threonine protein phosphatase-5 (PP5) is involved in tumor progression and survival, making it an attractive therapeutic target. Specific inhibition of protein phosphatases has remained challenging because of their conserved catalytic sites. PP5 contains its regulatory domains within a single polypeptide chain, making it a more desirable target. Here we used an in silico approach to screen and develop a selective inhibitor of PP5. Compound P053 is a competitive inhibitor of PP5 that binds to its catalytic domain and causes apoptosis in renal cancer. We further demonstrated that PP5 interacts with FADD, RIPK1, and caspase 8, components of the extrinsic apoptotic pathway complex II. Specifically, PP5 dephosphorylates and inactivates the death effector protein FADD, preserving complex II integrity and regulating extrinsic apoptosis. Our data suggests that PP5 promotes renal cancer survival by suppressing the extrinsic apoptotic pathway. Pharmacologic inhibition of PP5 activates this pathway, presenting a viable therapeutic strategy for renal cancer.

Proteostasis, the maintenance of proper protein folding, stability, and degradation within cells, is fundamental for cellular function. Two key players in this intricate cellular process are macroautophagy/autophagy and chaperoning of nascent proteins. Here, we explore the crosstalk between autophagy and the HSP90 chaperone in maintaining proteostasis, highlighting their interplay and significance in cellular homeostasis. Abbreviation: HSP90: heat shock protein 90; PTMs: post-translational modifications [ABSTRACT FROM AUTHOR]

Cellular homeostasis relies on both the chaperoning of proteins and the intracellular degradation system that delivers cytoplasmic constituents to the lysosome, a process known as autophagy. The crosstalk between these processes and their underlying regulatory mechanisms is poorly understood. Here, we show that the molecular chaperone heat shock protein 90 (Hsp90) forms a complex with the autophagy-initiating kinase Atg1 (yeast)/Ulk1 (mammalian), which suppresses its kinase activity. Conversely, environmental cues lead to Atg1/Ulk1-mediated phosphorylation of a conserved serine in the amino domain of Hsp90, inhibiting its ATPase activity and altering the chaperone dynamics. These events impact a conformotypic peptide adjacent to the activation and catalytic loop of Atg1/Ulk1. Finally, Atg1/Ulk1-mediated phosphorylation of Hsp90 leads to dissociation of the Hsp90:Atg1/Ulk1 complex and activation of Atg1/Ulk1, which is essential for initiation of autophagy. Our work indicates a reciprocal regulatory mechanism between the chaperone Hsp90 and the autophagy kinase Atg1/Ulk1 and consequent maintenance of cellular proteostasis. [Display omitted] • Autophagy-initiating kinase Atg1/Ulk1 phosphorylates a conserved serine in Hsp90 • Atg1/Ulk1-mediated phosphorylation of Hsp90 is essential for autophagy induction • Hsp90 phosphorylation by Atg1/Ulk1 regulates client protein activity • Phosphorylation of Hsp90 is essential for Atg1 conformational change and activity Backe et al. show a reciprocal regulatory mechanism between the chaperone Hsp90 and the autophagy kinase client Atg1/Ulk1. This consequently leads to maintenance of cellular proteostasis and activation of autophagy. [ABSTRACT FROM AUTHOR]

Heat shock protein-90 (Hsp90) chaperone machinery is involved in the stability and activity of its client proteins. The chaperone function of Hsp90 is regulated by co-chaperones and post-translational modifications. Although structural evidence exists for Hsp90 interaction with clients, our understanding of the impact of Hsp90 chaperone function toward client activity in cells remains elusive. Here, we dissect the impact of recently identified higher eukaryotic co-chaperones, FNIP1/2 (FNIPs) and Tsc1, toward Hsp90 client activity. Our data show that Tsc1 and FNIP2 form mutually exclusive complexes with FNIP1, and that unlike Tsc1, FNIP1/2 interact with the catalytic residue of Hsp90. Functionally, these co-chaperone complexes increase the affinity of the steroid hormone receptors glucocorticoid receptor and estrogen receptor to their ligands in vivo. We provide a model for the responsiveness of the steroid hormone receptor activation upon ligand binding as a consequence of their association with specific Hsp90:co-chaperone subpopulations. [Display omitted] • FNIP1 forms mutually exclusive functional complexes with FNIP2 and Tsc1 co-chaperones • Hsp90 co-chaperone complexes differentially regulate client protein activity • FNIP1/2 and Tsc1 enhance steroid hormone receptor ligand binding and activity • Hsp90 induces structural changes in the glucocorticoid receptor ligand-binding pocket Backe et al. demonstrate that responsiveness of the steroid hormone receptors upon ligand-binding-mediated activation depends on their association with specific Hsp90:co-chaperone (FNIP1, FNIP2, and Tsc1) subpopulations. This study reveals that formation of different Hsp90:co-chaperone:client complexes provide differentially primed pools of client proteins ready to act in different cellular environments. [ABSTRACT FROM AUTHOR]

The extracellular molecular chaperone heat shock protein 90 (eHSP90) stabilizes protease client the matrix metalloproteinase 2 (MMP2), leading to tumor cell invasion. Although co-chaperones are critical modulators of intracellular HSP90:client function, how the eHSP90:MMP2 complex is regulated remains speculative. Here, we report that the tissue inhibitor of metalloproteinases-2 (TIMP2) is a stress-inducible extracellular co-chaperone that binds to eHSP90, increases eHSP90 binding to ATP, and inhibits its ATPase activity. In addition to disrupting the eHSP90:MMP2 complex and terminally inactivating MMP2, TIMP2 loads the client to eHSP90, keeping the protease in a transient inhibitory state. Secreted activating co-chaperone AHA1 displaces TIMP2 from the complex, providing a "reactivating" mechanism for MMP2. Gene knockout or blocking antibodies targeting TIMP2 and AHA1 released by HT1080 cancer cells modify their gelatinolytic activity. Our data suggest that TIMP2 and AHA1 co-chaperones function as a molecular switch that determines the inhibition and reactivation of the eHSP90 client protein MMP2. • Stress-inducible TIMP2 is a bona fide co-chaperone of extracellular HSP90 (eHSP90) • TIMP2 regulates HSP90 chaperone function and interaction with client MMP2 • Secreted co-chaperones TIMP2 and AHA1 displace each other on the eHSP90:MMP2 complex • TIMP2-AHA1 competition impacts client MMP2 activity and matrix gelatinolysis Hundreds of intracellular proteins depend on molecular chaperone heat shock protein 90 (HSP90) and its co-chaperones to properly function. Baker-Williams et al. identify two secreted co-chaperones that act as a molecular switch to inhibit and reactivate extracellular HSP90 (eHSP90) client matrix metalloproteinase 2 (MMP2). This mechanism impacts matrix degradation. [ABSTRACT FROM AUTHOR]

Summary The molecular chaperone Hsp90 stabilizes and activates client proteins. Co-chaperones and post-translational modifications tightly regulate Hsp90 function and consequently lead to activation of clients. However, it is unclear whether this process occurs abruptly or gradually in the cellular context. We show that casein kinase-2 phosphorylation of the co-chaperone folliculin-interacting protein 1 (FNIP1) on priming serine-938 and subsequent relay phosphorylation on serine-939, 941, 946, and 948 promotes its gradual interaction with Hsp90. This leads to incremental inhibition of Hsp90 ATPase activity and gradual activation of both kinase and non-kinase clients. We further demonstrate that serine/threonine protein phosphatase 5 (PP5) dephosphorylates FNIP1, allowing the addition of O -GlcNAc (O -linked N-acetylglucosamine) to the priming serine-938. This process antagonizes phosphorylation of FNIP1, preventing its interaction with Hsp90, and consequently promotes FNIP1 lysine-1119 ubiquitination and proteasomal degradation. These findings provide a mechanism for gradual activation of the client proteins through intricate crosstalk of post-translational modifications of the co-chaperone FNIP1. Graphical Abstract Highlights • Casein-kinase-2-mediated sequential phosphorylation of the co-chaperone FNIP1 • FNIP1 relay phosphorylation leads to gradual activation of Hsp90 clients • Serine/threonine protein phosphatase 5 (PP5) dephosphorylates FNIP1 • O -GlcNAcylation causes ubiquitination and proteasomal degradation of FNIP1 Sager et al. show that casein-kinase-2-mediated sequential phosphorylation of the co-chaperone FNIP1 leads to incremental inhibition of Hsp90 ATPase activity and gradual activation of both kinase and non-kinase clients. O -GlcNAcylation antagonizes phosphorylation of FNIP1, preventing its interaction with Hsp90, and consequently promotes FNIP1 ubiquitination and proteasomal degradation. [ABSTRACT FROM AUTHOR]

New roles for Tsc1 and FNIP1/2 as regulators of the molecular chaperone Hsp90 were recently identified, demonstrating a broader cellular impact outside of AMPK-mTOR signaling. In studying the function of these proteins we must take a holistic view of the cell, instead of maintaining our focus on a single pathway. [ABSTRACT FROM AUTHOR]